Interpretive Summary: The first two human illness outbreaks attributed to consumption of ground beef contaminated with Escherichia coli O157 occurred in 1982. Now cases of human foodborne E. coli O157 are reported nearly every day in US news. Estimates suggest the total cost of health care to treat these infections is $1-2 billion with 500 deaths per year. Although in recent years E. coli O157 outbreaks have been traced to various foods, ground beef remains a major source. The bacterium causes disease by producing toxins, including Shiga toxins 1 and 2. It may be detected by identification of these two toxins as well as the O157 “signature” molecule on the surface of the bacteria. However no single commercial test is capable of simultaneous detection of E. coli O157 and the toxins. Thus we developed a new test that can simultaneously detect O157 and its toxins in ground beef in less than 24 hours. The test works by capturing live E. coli O157 onto fluorescent microbeads. Because the microbeads are also magnetic, our test simplifies recovery of the bacteria from ground beef. This feature is attractive to regulatory agencies (e.g., the US Food and Drug Administration and the USDA Food Safety and Inspection Service), whose policies require isolation of the disease-causing bacteria from food, before they can take action, such as a product recall. Moreover, our test may be used in meat processing plants to allow in-house testing of products prior to sale. This would reduce the frequency of foodborne illness, reduce product recalls, and enhance public health while reducing annual cost for food testing.

Technical Abstract:
Shiga toxin-producing Escherichia coli (STEC) are costly foodborne pathogens. As the global prevalence of STEC in beef products ranges from 0.01% to 62.5%, there is a need for developing a rapid screening assay. Here, we developed a Luminex immunoassay to screen for Shiga toxin 1 (Stx1), Shiga toxin 2, and E. coli O157 lipopolysaccharides (O157 LPS) simultaneously in ground beef. Using minimal sample preparation, we tested ground beef samples spiked with < 50 CFU of STEC organisms for Stx1, Stx2, and O157 LPS. We were able to detect the three analytes simultaneously and our results showed the same specificity and sensitivity as ones obtained from testing pure STEC cultures. Conventional sandwich ELISA using the same antibodies was not as sensitive. Our assay serves as a milestone for developing a multiplex assay for additional foodborne pathogens, as the Luminex technology may be used for up to 100 distinct simultaneous reactions.